PROJECT SUMMARY Castration-resistant prostate cancer (CRPC) is the major cause of prostate cancer (PCa) mortality. Targeting the androgens-androgen receptor (AR) axis by the second-generation endocrine therapies such as abiraterone (ABI) and enzalutamide (ENZ) has been effective initially for CRPC treatment in clinic. However, most patients develop resistance that undermines survival and quality of life and the therapy resistance is likely due to expression of AR splice variants (AR-Vs) such as AR-V7 and/or other undefined mechanisms. Prostate- specific antigen (PSA or known as KLK3) is one of the genes that are highly transcriptionally activated by AR. Despite being extensively utilized as a biomarker for PCa diagnosis and prognosis, the significance of the PSA gene or the entire genomic locus of this gene in PCa growth and survival has yet been well established. We demonstrated that non-coding RNA transcribed from the PSA enhancer, or called PSA eRNA, is aberrantly upregulated in CRPC cells in culture, xenografts and patient tissues. We further showed that expression of PSA eRNA is regulated by AR-Vs in ENZ-resistant CRPC cells. Moreover, we showed that PSA eRNA has cis-effects on PSA mRNA expression as well as broad trans-effects on expression of AR-regulated biologically important genes in CRPC cells. Mechanistically, we found that PSA eRNA contains a HIV-1 TAR RNA like (TAR-L) motif that is crucial for binding to CYCLIN T1, a key component of the positive transcription elongation factor b (P-TEFb) complex and P-TEFb-mediated RNA polymerase II serine 2 phosphorylation (Pol II-Ser2p). Importantly, we demonstrated that targeting PSA eRNA with generation 2.5 antisense oligonucleotides (ASOs) inhibits growth of ENZ-resistant CRPC cells. Our further studies showed that eRNA transcribed from the FTO gene enhancer, one of the AR-eRNAs induced by ENZ, binds to RNA splicing factors and regulates expression of AR splice variant AR-V7 in ENZ-resistant CRPC cells. These findings support the hypothesis that aberrant expression of AR-eRNAs including PSA and FTO eRNAs acts as a new proxy of AR functional abnormality that promotes endocrine therapy-resistant growth of CRPC, thereby representing a novel target for CRPC treatment. In this application, we will determine the mechanisms by which PSA eRNA regulates gene transcription and FTO eRNA regulates AR-V7 expression in ENZ-resistant CRPC cells (Aim 1) and to determine the functional importance and clinical significance of PSA eRNA expression in ENZ- and ABI- resistant CRPC cells using mouse models and human patient samples (Aim 2). These studies will employ a comprehensive approach bringing complementary expertise in tumor biology, molecular biology, medical oncology, pathobiology, computing science and bioinformatics and biomedical statistics. Findings from the proposed studies will significantly advance our understanding of the mechanisms that drives aberrant AR activity and therapy-resistant growth of CRPC cells, but also allow us to develop new strategies by targeting PSA eRNA to inhibit aberrant AR activity for effective treatment of CRPC.